N-Acetyl Lysyltyrosylcysteine Amide Inhibits Myeloperoxidase, a Novel Tripeptide Inhibitor Hao Zhang1,5, Xigang Jing 1,5, Yang Shi1,5,8, Hao Xu1,5, Jianhai Du1,5, Tongju Guan1,5, Dorothee Weihrauch3, Deron W. Jones1,5, Weiling Wang6,7, David Gourlay1,5, Keith T. Oldham1,5, Cheryl A. Hillery2,4, and Kirkwood A. Pritchard Jr.1,5 Departments of 1Surgery, Division of Pediatric Surgery; 2Departments of Pediatrics, Division of Hematology/Oncology and 3Anesthesiology;
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Blood Research Institute of Wisconsin;
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Children’s Research Institute, Medical College of Wisconsin; 6Department of Geriatrics, Qilu
Hospital, Shandong University; 7Key Laboratory of Cardiovascular Proteomics of Shandong
Mailing address: 1, 2, 3, 4 6, 7 8
and 5 8701 Watertown Plank Road, Milwaukee, WI, 53226
Jinan, Shandong, 250012 People’s Republic of China
1020 North 12th Street, Suite 401, Milwaukee, WI 53233
Corresponding Authors: Hao Zhang, Ph. D Children’s Research Institute C4415 Medical College of Wisconsin 8701 Watertown Plank Road Milwaukee, WI 53226 Office: 414-955-5807 Fax: (414) 955-6473 e-mail:
[email protected]
Kirkwood A. Pritchard Jr., Ph.D. Children’s Research Institute C4420 Medical College of Wisconsin 8701 Watertown Plank Road Milwaukee, WI 53226 Office: 414-955-5615 Fax: (414) 955-6473 e-mail:
[email protected]
Running title: Novel Tripeptide Inhibitors of Myeloperoxidase Activity Abbreviations: KYC, N-acetyl lysyltyrosylcysteine amide; HOCl, hypochlorous acid; O2•─, superoxide anion; Tyr, tyrosine; Trp, tryptophan; MPO, myeloperoxidase; yNO2, nitrogen dioxide radical; ClTyr, chlorotyrosine; Cys, cysteine; DiTyr, dityrosine; BAEC, bovine aortic endothelial cells; HL-60, human promyelocytic leukemia cells; DTPA, diethylene triamine pentaacetic acid; NO2Tyr, nitrotyrosine; MDA, malondialdehyde. Support: Supported by AHA 11SDG5120015 (H. Z), R01-HL089779 (DW), R01-HL102836 (KAP& CAH), R21-HL102996 (KAP), U54-HL090503 (CAH) and a generous gift from Ms. Poblocki of Elm Grove, WI (KAP).
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Province; 8Patient Centered Research, Aurora Health Care, Milwaukee, Wisconsin.
Abstract Myeloperoxidase (MPO) plays important roles in disease by increasing oxidative and nitrosative stress and oxidizing lipoproteins. Here we report N-acetyl lysyltyrosylcysteine amide (KYC) is an effective inhibitor of MPO activity.
We show KYC inhibits MPO-mediated
hypochlorous acid (HOCl) formation and nitration/oxidation of LDL.
Disulfide is the major
product of MPO-mediated KYC oxidation. KYC (≤4000 µM) does not induce cytotoxicity in bovine aortic endothelial cells (BAEC). KYC inhibits HOCl generation by phorbol myristate acetate (PMA)-stimulated neutrophils and HL-60 cells but not superoxide generation by PMAKYC inhibits MPO-mediated HOCl formation in BAEC culture and
protects BAEC from MPO-induced injury. KYC inhibits MPO-mediated lipid peroxidation of LDL whereas tyrosine (Tyr) and tryptophan (Trp) enhance oxidation. KYC is unique as its isomers do not inhibit MPO activity, or are much less effective. UV-Vis spectral studies indicate KYC binds to the active site of MPO and reacts with compound I and II. Docking studies show the tyrosine of KYC rests just above the heme of MPO.
Interestingly, KYC increases MPO-
dependent H2O2 consumption. These data indicate KYC is a novel and specific inhibitor of MPO activity that is nontoxic to endothelial cell cultures.
Accordingly, KYC may be useful for
treating MPO-mediated vascular disease. Key words: Myeloperoxidase, lipid peroxidation, hypochlorous acid, nitrogen dioxide, KYC, inhibitor, nitration, chlorination, low density lipoproteins, Apolipoportein A1
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stimulated HL-60 cells.
Introduction Myeloperoxidase (MPO) is a heme peroxidase released from activated neutrophils, macrophages and monocytes that plays important roles in host defense (1-3). Ferric MPO reacts with hydrogen peroxide (H2O2) to form compound I, an oxy-ferryl-cation radical (P•Fe4+=O) intermediate. This intermediate can oxidize a wide variety of substrates to generate an equally wide variety of toxic oxidants and free radicals to kill invading bacteria. Compound I oxidizes (pseudo)halides (such as chloride (Cl¯), bromide (Br¯) and thiocynate (SCN¯)) via direct, two-electron reduction (halogenation cycle) to form corresponding (pseudo)hypohalous
(HOSCN)). MPO oxidizes organic substrates such as Tyr and Trp to form tyrosyl (Tyry) and tryptophanyl (Trpy) radicals, respectively. MPO also oxidizes a wide variety of ionic species (nitrite (NO2¯), ascorbate and urate) via one-electron reduction (peroxidation cycle) to form free radicals (nitrogen dioxide radicals (yNO2), ascorbyl radicals and urate radicals) (4, 5). Although MPO is released as a means of killing invading bacteria, activated immune cells have been reported to release MPO even in the absence of infection, which unfortunately induces vascular injury and damage (2, 6, 7). Growing evidence supports the idea that MPO plays important roles in the pathogenesis of disease by increasing oxidative and nitrosative stress (6). Oxidative stress induced by aberrant MPO activity has been observed in inflammatory lung disease (8), rheumatoid arthritis (9, 10), peripheral artery disease (11), cardiovascular disease (7, 12) and diabetes (13, 14). Even basic science studies in rats have shown that MPO directly correlates with severity of myocardial infarction (15, 16). Recently, immunochemical studies revealed that MPO is expressed in microglia, astrocytes and certain types of neurons, suggesting that MPO could play an important role in neurodegenerative disease (17), such as multiple sclerosis (18-20), Alzheimer’s (21, 22) and Parkinson’s disease (23). Interestingly, MPO has even been implicated as a risk factor for some forms of cancers (24, 25).
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Some of
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acids (such as hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocynate
the earliest evidence that MPO plays a role in cardiovascular disease comes from studies showing that chlorotyrosine (ClTyr) on LDL is increased in human vascular lesions (36). More recently, several groups have suggested that MPO oxidation of HDL may also play a role in atherosclerosis (26-29). With such growing evidence that MPO plays a causal role in a variety of diseases, it seems important to develop an inhibitor that can be used to prevent MPOdependent oxidative damage (30). A variety of different approaches have been used to inhibit MPO-mediated cell injury (31): antioxidant scavenging of MPO oxidants/radicals; inhibiting H2O2 production in vivo; and, Antioxidant scavenging of MPO oxidants and free radicals
turned out to be an ineffective approach because the reaction between MPO oxidants (i.e., HOCl, HOBr) and antioxidants was not fast enough to prevent tissue damage (32-34). Inhibiting cell injury by MPO via blocking H2O2 production in vivo was also considered impractical because multiple pathways exist for generating H2O2 and none of the agents were able to block H2O2 from all sources (35). Although suicide inhibitors (i.e., azides, hydrazides and hydroxamic acids) that irreversibly modify the iron heme site of MPO, are highly effective for inhibiting enzyme activity in vitro (31), they lack specificity and are inherently toxic, which makes them undesirable as therapeutic agents (35). Several indole derivatives have been used as reversible inhibitors of MPO because they effectively compete with Cl¯ and SCN¯ to prevent Compound I from generating HOCl and HOSCN (36, 37). However, during oxidation, these agents are converted into radicals that are toxic and capable of increasing oxidative stress in vivo (36, 38, 39). Phenolic compounds have also been used to inhibit MPO because they compete with the other substrates for both compound I and II (35, 40, 41). However, MPO oxidization of phenolic compounds also results in the formation of toxic radicals that can increase oxidative stress. For example, MPO has been shown to oxidize several phenolic compounds into radicals that
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directly inhibiting MPO activity.
actually accelerate LDL oxidation (42-44). A significant amount of effort has gone into designing and testing agents that block MPO activity. Recent reports show that 2-thioxanthine and INV315 inhibit MPO in vivo (45, 46). It is well-known that MPO oxidizes the phenol side-chain of tyrosine in small peptides(47, 48) and it generates oxidants that oxidize large proteins to form nitrotyrosine and/or dityrosine (DiTyr) adducts (49). Here, we explored the possibility of using a series of novel tripeptides containing both Tyr and cysteine (Cys) as MPO inhibitors, whereby a Tyry formed by MPO activity is scavenged by the thiol of the adjacent Cys (48, 50, 51). In this way the ability of
Our studies show that KYC inhibits MPO-dependent HOCl generation, protein nitration and LDL oxidation. Further, KYC specifically inhibits MPO and induces little if any cytotoxicity making it highly effective for protecting cells from MPO-induced injury.
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Tyry to leave the active site and oxidize LDL and/or induce cytotoxicity is essentially eliminated.
Materials and Methods Materials: MPO and LDL were from Lee Biosolutions (St. Louis, MO). Catalase, superoxide dismutase and rabbit anti-NO2Tyr polyclonal antibody were from EMD (Gibbstown, NJ). MPO antibody was from Calbiochem (Cambridge, MA). KYC and other tripeptide analogs were either synthesized by the Blood Center of Wisconsin (Milwaukee, WI) or Biomatik (Wilmington, DE). All other chemicals and reagents were from Sigma-Aldrich (St. Louis, MO). Purity (>98%) and authenticity of the tripeptides were confirmed by HPLC analysis and mass spectrometry. HL-60 cells (human promyelocytic leukemia cells) were from American Type Culture Collection (ATCC) BAEC were obtained and maintained as previously described (52). The
Homogeneous Caspases Assay kit (catalog No. 03005372001) was from Roche (Indianapolis, IN). CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit (Catalog No. G3580) and Mitochondrial ToxGlo™ Assay kit (Catalog No. G8000) were from Promega (Madison, WI). MPO-catalyzed HOCl Production: MPO (20 nM) was incubated with H2O2 (50 µM), NaCl (150 mM), taurine (5 mM) and increasing concentrations of KYC in a phosphate buffer (100 mM, pH 7.4) containing diethylene triamine pentaacetic acid (DTPA, 100 µM) to prevent non-specific, divalent metal cation oxidation for 30 min. Reactions were halted by addition of catalase (2,000 units/mL). Taurine chloramine was quantified using the TMB assay (53). Briefly, 400 µL of reaction solution was mixed with 100 µL of 2 mM TMB, 100 µM NaI containing 10% dimethylformamide (DMF) in 400 mM acetate buffer (pH 5.4). After 5 min, absorbance (650 nm) was recorded on a UV-Vis spectrophotometer (Agilent Model 8453). MPO-mediated LDL Conjugated Diene Formation:
Reaction mixtures contained LDL (0.15
mg/mL), NaNO2 (100 μM), H2O2 (100 μM), MPO (20 nM) and increasing concentrations of KYC or equimolar concentrations of various compounds in a phosphate buffer (100 mM, pH 7.4) containing DTPA (100 μM). Rates of LDL conjugated diene formation were determined by
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(Manassas, VA).
following changes in absorbance at 234 nm, the absorption maximum for conjugated dienes, on a UV-Vis spectrophotometer (Agilent Model 8453) at room temperature. MPO-mediated LDL Malondialdehyde (MDA) Formation: Reaction mixtures contained LDL (0.5 mg/mL), NaNO2 (50 μM), H2O2 (50 μM), MPO (50 nM) and increasing concentrations of KYC in a phosphate buffer (100 mM, pH 7.4) containing DTPA (100 μM). After incubation at 37 ºC for 4 h, the reactions were stopped by addition of catalase (2,000 units/mL). The formation of MDA was determined according to published procedures (54, 55).
Briefly, incubation mixtures
(containing 25 mM BHT) were adjusted to pH 1.5 and incubated at 60 ºC for 80 min to hydrolyze
N-methyl-2-phenylindole (13.4 mM in acetonitrile/methanol (3:1)). After centrifugation (13,000 x g, 5 min), 330 µL of the supernatants were mixed with 57.5 µL of concentrated HCl and incubated at 45 ºC for another 60 min. Finally, after centrifugation (13,000 x g, 5 min), total MDA in the samples was determined from the absorbance at 586 nm using a UV-Vis spectrophotometer (Agilent Model 8453). MPO-mediated LDL Trp Oxidation: Reaction mixtures containing LDL (0.15 mg/mL), NaNO2 (100 μM), H2O2 (100 μM), MPO (20 nM) and increasing concentrations of KYC in a phosphate buffer (100 mM, pH 7.4) containing DTPA (100 μM) were incubated at room temperature for 30 min. Reactions were stopped by addition of catalase (2,000 units/mL) and the oxidation of Trp in LDL was determined by measuring changes in the intrinsic fluorescence of Trp (Ex 294nm/Em 345 nm) using a LC-50 fluorometer (Perkin Elmer, Waltham, MA). MPO-mediated Nitration of LDL: LDL (0.5 mg/mL) was incubated with MPO (50 nM), H2O2 (50 µM), NaNO2 (50 µM) and increasing concentrations of KYC in phosphate buffer (100 mM, pH 7.4) containing DTPA (100 µM) at 37 ºC for 4 h.
Reactions were stopped by addition of
catalase (2,000 units/mL). Formation of NO2Tyr was assessed by dot blot analysis. Briefly, LDL solutions were mixed with 1% SDS and centrifuged (12,000 x g, 15 min).
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the Schiff Bases formed from MDA and protein. The samples were mixed with 3-fold volume of
supernatants were applied to a nitrocellulose membrane using a dot blot apparatus (Bio-Rad model Bio-Dot). The levels of NO2Tyr were visualized using a rabbit polyclonal anti NO2Tyr antibody (EMD, Gibbstown, NJ) and the ECL plus kit from Thermo-Peirce (Rockford, IL). HPLC Analysis: KYC oxidation products were analyzed by reverse phase HPLC using a C-18 column (4.6x150 mm). The peptide and products were eluted using an acetonitrile gradient (5 10%, containing 0.1% trifluoroacetic acid) for 20 min. Elution was monitored at both 220 nm and 280 nm. N-Acetyl lysyltyrosylserine amide (KYS) and N-acetyl lysylphenylalanylcysteine amide (KFC) were analyzed on a C-18 column (2.2x150 mm) and eluted with an acetonitrile
Cytotoxicity Assays. BAEC (passage 4-10) were seeded onto 96-well plates and cultured in MEM medium containing 10% FBS in a 5% CO2 and 100% humidity environment at 37 ºC. Increasing concentrations of KYC (0 to 4mM, final concentration) were added to the culture medium and cells were incubated for another 24 h. The effects of KYC on cell viability were determined by the MTS assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit, Promega, Madison, WI). Caspase activities for apoptosis in the treated BAEC were measured with the Homogeneous Caspases Assay kit from Roche (Indianapolis, IN).
Necrosis and
mitochondrial functions were analyzed by Mitochondrial ToxGlo™ Assay kit (Promega, Madison, WI). All determinations were performed according to manufacturer’s instructions. Phorbol 12-myristate 13-acetate (PMA)-induced HOCl Formation by HL-60 cells: HL-60 cells were cultured in RPMI 1640 medium containing 10% FBS (passage 20-50).
Cells were
harvested by centrifugation (1000 rpm, 10 min) and washed twice with Dulbecco’s phosphate buffered saline (DPBS) with glucose. HL-60 cells (1.2 x107/mL) were re-suspended in DPBS with glucose. The washed HL-60 cells were either stimulated with PMA (10 µM) or not, and incubated with taurine (5 mM) and increasing concentrations of KYC at 37 ºC for 30 min.
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gradient (5 - 30%, containing 0.1% trifluoroacetic acid) for 25 min.
Catalase (2,000 units/mL) was added to halt the reaction. After centrifugation, the supernatants were analyzed using the TMB assay as outlined above. Phorbol 12-myristate 13-acetate (PMA)-induced HOCl Formation by Human Neutrophils: Human neutrophils were isolated according to a previous report (56). All protocols utilizing human neutrophils were approved by the Medical College of Wisconsin Institutional Review Board. The KYC inhibition of HOCl formation from PMA-stimulated neutrophils was analyzed as described in reference (57). Briefly, neutrophils (3 x106 cells/mL) were mixed with different amounts of KYC in Hank’s balance salt solution containing MgCl2 (0.5 mM), CaCl2 (1.26 mM),
incubated at 37 ºC for 20 min. the reactions were stopped by catalase (2,000 units/mL). After centrifugation, the supernatants were analyzed using the TMB assay as previously described. PMA-stimulated HL-60 Cell O2•─ Formation: HL-60 cells were harvested by centrifugation at 1,000 rpm for 10 min), washed twice with DPBS with glucose to remove culture medium. The HL-60 cells (1.2 x107/mL) were re-suspended in DPBS with glucose. After stimulation with PMA (10 µM), the HL-60 cells were incubated with Cytochrome C (40 µM) with or without superoxide dismutase (500 units/mL) at 37 ºC for 10 min and then the HL-60 cells were removed by centrifugation. Cytochrome C reduction in supernatants was measured at 550 nm using a UVVis spectrophotometer (Agilent Model 8453). MPO-mediated BAEC Injury: BAEC (passage 6-8) were cultured in 24-well plates in DMEM containing 10% FBS until 70-80% confluent. The cells were washed with Hank’s balanced salt solution (HBSS) 3 times and then incubated with MPO (2.5 μg/mL) and H2O2 ± KYC at the concentrations indicated in HBSS (0.5 ml) at 37 °C for 30 min. In the case of neutrophils as a source of both MPO and H2O2, neutrophils (0.2 x106 cells/0.5 mL) were added into 24 well plates in the presence of different amounts of KYC in HBSS with MgCl2, CaCl2 and glucose. The cells were stimulated with 2 µM PMA at 37 ºC for 30 min. The cultured cells were washed
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glucose (5.5 mM) and taurine (5 mM). The cells were stimulated with PMA (100 ng/mL) and
with HBSS 3 times. Finally, BAEC were examined visually and images captured for permanent record as described (58) using a Nikon Eclipse microscope (Model TS100) fitted with a Nikon Digital Sight DS-U2 camera and NIS-Elements F 3.0 imaging software.
Images are
representative of 3 independent experiments. Effects of KYC on MPO UV-Vis Spectra: MPO (1.4 μM) was incubated with KYC (50 μM) with or without H2O2 (40 μM) and NaCl (150 mM) at room temperature. The changes in UV-Vis spectra were recorded as indicated. Compound II was prepared by mixing MPO (1.4 μM) with H2O2 (300 μM) for 20 sec. Excess H2O2 was removed by addition of catalase (5 µg/mL). The
recorded. Experiments were also performed in the presence of methionine (1 mM). The results show no difference in UV-Vis spectra in the presence or absence of methionine. Statistics: Data are presented as mean ± SD unless stated otherwise and analyzed with the student’s t-test where appropriate using Prism 5.0 (Graph Pad, Inc.) for two group comparison.
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reaction was immediately mixed with KYC (50 μM) and the changes in heme spectra were
Results Effects of Tripeptides on MPO-catalyzed HOCl Production: To determine the extent to which tripeptides inhibit MPO activity, we synthesized a series of 6 tripeptides containing Tyr and Cys (XYC) and studied their effects on MPO HOCl generation. As shown in Figure 1A, at 12.5 μM, among the tripeptides tested, KYC was the only tripeptide that reduced HOCl production by ~75%. To exclude the possibility that such reduction is due to a direct scavenging of HOCl or taurine chloramine by KYC, we mixed KYC with HOCl or preformed taurine chloramine and analyzed the remaining HOCl or taurine chloramine by TMB/KI assay. Briefly, KYC [6.25 µM
chloramine under our assay conditions for 30 min, then measurements of HOCl or taurine chloramine were performed as described in Methods. Our data shows that one molecule of KYC scavenges 1.21 ± 0.15 (n=6) HOCl molecules or 0.97 ± 0.01 (n=6) molecules of taurine chloramine. No significant differences were noted between the two KYC concentrations used (6.25 µM and 12.5 µM). These results suggest that KYC reduces HOCl formation by inhibiting MPO activity, not just scavenging HOCl or taurine chloramine. The other tripeptides scavenge HOCl production by (~27% to ~0%). These data indicate that although KYC scavenges HOCl to the same extent as other thiol peptides, significantly greater inhibition is achieved when it is treated with MPO that cannot be explained as scavenging HOCl.
KYC’s ability to inhibit MPO
is comparable to the ability of Trp to inhibit MPO (36). Interestingly, when arginine, another positively charged amino acid, is substituted for Lys the ability of RYC to inhibit MPO-catalyzed HOCl production is markedly reduced (KYC = ~75% vs. RYC = ~22% inhibition). These data indicate that the charge, size and hydrophobicity of the first amino acid are all important properties for how tripeptides inhibit MPO activity. To further assess the efficiency of KYC for inhibiting MPO, we next determined dosedependent effects of KYC on MPO-catalyzed HOCl production. Figure 1B shows that KYC
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(n=3) and 12.5 µM KYC (n=3)] was incubated with either 50 µM HOCl or 50 µM taurine
dose-dependently inhibited HOCl production with an IC50 of ~7 µM. At 25 µM, KYC completely inhibited HOCl production (Fig. 1B). To understand the importance of the phenol of Tyr and the thiol of Cys for KYC inhibiting MPO, we also compared the dose-dependent effects of KYC with two structural analogues, KFC and KYS. Without a free thiol, KYS failed to inhibit MPO-catalyzed HOCl production (Fig. 1C). Although KFC decreased the amount of HOCl detected by the taurine/TMB assay, its ability to decrease HOCl was much less than KYC (Fig. 1D).
Where KYC (25 µM) completely ablated
MPO-catalyzed HOCl production, KFC (25 µM) reduced HOCl by only 35 % (Fig. 1D). As both
MPO, it is likely that KFC’s mechanism of action has more to do with the free thiol scavenging than actually entering the active site of MPO and reacting compound I or II as does KYC. While Tyr and Cys are required for KYC to inhibit MPO, these data suggest that Lys also plays an important role in orienting the tripeptide for optimal inhibition. To investigate the effect of D-isomers and sequence isomers of KYC on MPO inhibition, we compared the effects of KYC, made with all L-amino acids, with the effects of D-amino acids and sequence isomers of KYC on MPO-catalyzed HOCl production. Figure 1E shows that replacing an L-amino acid with a D-amino acid at any position or even at all three positions in KYC dramatically decreased the ability of the tripeptide to inhibit MPO-catalyzed HOCl production. Likewise, YKC and CYK failed to inhibit MPO-catalyzed HOCl production to the same extent as KYC (all L-amino acids) (Fig. 1E). Finally, we compared the effects of KYC on MPO-catalyzed HOCl production to the effects of free Tyr and Trp (Fig. 1F). Consistent with another report (36), Trp was an effective inhibitor of MPO-catalyzed HOCl production.
In
contrast, Tyr alone had little, if any effect, on MPO-catalyzed HOCl production, as has been reported (36). The lack of effect of Tyr on MPO activity is also in agreement with data showing that KYS, which also contains a single Tyr, is not an effective inhibitor of MPO-catalyzed HOCl
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Phe and Cys are considered poor substrates for MPO and there is almost no direct oxidation by
production. These data indicate that KYC’s sequence is unique and that steric conformation and amino acid sequence order are important structural requirements for KYC to inhibit MPO. MPO Catalyzed KYC Oxidation Product Analysis. To determine how MPO oxidizes KYC, we analyzed reaction products by HPLC. MPO/H2O2 systems oxidized KYC essentially to a single product that eluted around 7.6 min (Fig. 2A, trace b). Although KYC can be oxidized by H2O2 directly, incubations with H2O2 alone yielded very little of the 7.6 min product (Fig. 2A, trace a), which has the same retention time as authentic KYC disulfide (Fig. 2A, trace e). Monitoring the eluate from the HPLC with a fluorescent detector (Ex=290nm/Em=410nm), the fluorescent
b). This lack of fluorescence rules out DiTyr as a major product of oxidation. When KYS is oxidized with the MPO/H2O2 system, several products were observed to elute between 7-10 min (Fig. 2C, trace a and b).
The major peak in this trace has a fluorescent profile that is
characteristic of DiTyr (Fig. 2D, trace b), suggesting that, unlike KYC, Tyr in KYS was oxidized by MPO to form Tyry, which in turn forms DiTyr. Incubation of KFC, another KYC analog, with the MPO/H2O2 system did not yield MPO-dependent oxidation products (Fig. 2E and 2F), although small amounts of disulfides could be observed, which is likely a result of slow oxidation of the thiols by H2O2. Such data clearly indicate that, although MPO is able to oxidize Tyr in both KYC and KYS directly, oxidation of the Tyr in KYS forms DiTyr products, whereas the Cys in KYC rapidly scavenges Tyry radical leading to the formation of a disulfide instead of DiTyr. Analysis of products from the MPO/H2O2/NO2¯ system (Fig. 2A, trace c) or the MPO/H2O2/Cl¯ system (Fig. 2A, trace d) shows that KYC disulfide is the major oxidation product of these systems. These data suggest that regardless of the conditions under which MPO oxidizes KYC, in the presence of NO2¯ or Cl¯, the major oxidation product is KYC disulfide, not DiTyr. Such findings clearly indicate that MPO directly oxidizes the Tyr in the tripeptides. This can only be
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characteristic of a dityrosine (DiTyr), showed no significant peak formation (Fig. 2B, trace a and
accomplished if KYC enters the active site of MPO to generate a Tyry radical that is subsequently detoxified by the free thiol of Cys with high efficiency. To confirm that KYC oxidation by MPO yields a simple disulfide, we reduced the product using simple thiols such as glutathione (GSH) and dithiothreitol (DTT). Figure 3 (trace b and c) shows that when KYC disulfide was incubated with GSH the KYC disulfide is completely reduced to its KYC monomer (Fig. 3, trace b). More so, DTT completely reduced KYC disulfide to its KYC monomer within 5 min (Fig. 3, trace c). These data demonstrate that oxidation of KYC results in the formation of simple disulfides that are easily regenerated to its active
KYC Specifically Inhibits MPO Activity from HL-60 Cells and Human Neutrophils. HL-60 cells were stimulated with PMA to induce the release of MPO and treated with KYC to determine its effects on HOCl production. Without PMA stimulation, HL-60 cells produced little, if any HOCl (Fig. 4A, inset). However, after PMA stimulation, HL-60 cells generated high levels of HOCl (Fig. 4A, inset). KYC dose-dependently inhibited HOCl production by PMA-stimulated HL-60 cells with an IC50 ~7 µM (Fig. 4A). MPO-mediated HOCl formation requires H2O2 that is derived from O2y¯ generated by NADPH oxidase (NOX). To determine whether KYC or KYC disulfide inhibited NOX O2y¯ generation in HL-60 cells, we quantified O2y¯ production using the Cytochrome C assay.
This is important because, if KYC inhibited NOX activity, it would
decrease H2O2 formation making it appear as if KYC inhibited MPO. Neither KYC nor KYC disulfide had any significant effect on O2y¯ production in PMA-stimulated HL-60 cells (Fig. 4B). HPLC analysis of the oxidation products revealed that KYC disulfide was the major product from PMA-stimulated HL-60 cells (Fig, 4C). Taken together, these data suggest that KYC inhibited MPO but not NOX activity and that KYC disulfide is the major oxidation product when activated HL-60 cells are incubated with KYC. Figure 4D shows that KYC inhibits neutrophil-mediated
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monomeric form with physiologically relevant concentrations of GSH.
HOCl generation. Adding KYC to the PMA stimulated neutrophils dose-dependently inhibit MPO mediated HOCl formation. Cytotoxicity of KYC on BAEC. As a first step towards determining whether KYC is suitable for in vivo treatments, we incubated BAEC cultures with increasing KYC concentrations (0-4 µM). After 24 hours, we analyzed the impact of KYC exposure on cell viability, apoptosis, necrosis and mitochondria function.
Figure S1A shows the effects of KYC on BAEC viability.
No
increases in cell death were induced by KYC even at 4000 µM. To assess the impact of KYC on BAEC apoptosis, we used the Homogeneous Caspases Assay kit from Roche to analyze
multiple activated caspases (caspase 2, 3, 6, 7, 8, 9, 10).
Caspase activity was essentially
unaltered in BAEC cultures incubated with increasing KYC concentrations (Fig. S1B).
These
data indicate that KYC does not induce apoptosis. Membrane integrity studies show that KYC had no effects on protease activity, an index of necrosis, in BAEC cultures after 24 h (Fig. S1C). Finally, cellular ATP, an index of mitochondrial function, was unaltered in BAEC cultures incubated with KYC (Fig. S1D). On the basis of data from these studies we conclude that KYC does not induce cell damage even at concentrations up to 4000 µM. Compared to the basal levels of caspase activity (in the group without KYC treatment), BAEC treated with high KYC concentrations tended to decrease caspase activity, which may indicate that KYC protects cells at high concentration through its free thiol antioxidant properties.
This may explain why
protease activity was also decreased in BAEC cultures incubated with 4000 µM KYC. KYC Protects BAEC from MPO-induced Injury. With data indicating that KYC is not toxic to BAEC cultures, we next determined if KYC protects BAEC from MPO-induced injury. BAEC in 96-well plates were treated with 100 µl of HBSS containing MPO (2.5 µg/mL) and H2O2 (50 µM) for 30 min with or without KYC. Changes in cell morphology were recorded as a direct measure of injury as previously reported by others (58). Figure 5 shows that KYC dramatically increased
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changes in the activity of multiple capases in BAEC cultures since this kit is able to detect
BAEC viability and survival. Incubation of BAEC in the MPO/H2O2/Cl¯ alone caused severe cell damage as demonstrated by dramatic changes in cell morphology (Fig. 5A).
KYC dose-
dependently increased protection of BAEC cultures from MPO-induced injury (Fig. 5A). At 50 µM KYC, BAEC cultures had almost the same morphology as cultures that were not exposed to MPO (control). KYC also protected BAEC from even higher concentrations of H2O2 (100 µM) and MPO (5 µg/mL) (Fig. 5B and 5C). This study shows that KYC is fully capable of protecting BAEC from the cytotoxic effects of MPO. Incubation of PMA stimulated neutrophils with BAEC also induced BAEC damage (Fig. 5D) as did incubation with MPO/H2O2 (Fig. 5A).
KYC
(Fig. 5D). To examine these observations from a different perspective, we determined if KYC can inhibit MPO HOCl formation even in the presence of BAEC. Figure S2A shows that KYC dosedependently inhibits HOCl formation by MPO (2.5 µg/ml) and H2O2 (50 µM) in the presence of BAEC. KYC inhibits HOCl formation even when MPO and H2O2 are increased (5 µg/ml and 100 µM, respectively) (Fig S2B).
PMA-stimulated neutrophils generate HOCl when incubated with
BAEC cultures (Fig. S2C). KYC inhibits HOCl formation by PMA-activated neutrophils in the presence of BAEC. To determine the effects of KYC on cell viability we also performed the MTS assay under these same conditions.
As anticipated, KYC protected BAEC in the presence of
MPO/H2O2 (Fig. S3A and S3B) and PMA-stimulated neutrophils (Fig. S3C). These data confirm that KYC inhibits MPO/neutrophil dependent HOCl generation even in the presence of BAEC, which begins to explain why KYC can protect BAEC from the harmful effects of MPO activity. Effects of KYC on MPO-mediated LDL Lipid Peroxidation. MPO oxidation of LDL and HDL has been hypothesized to play a causal role in the genesis of atherosclerotic lesions. To determine if KYC inhibits LDL oxidation, we incubated LDL in MPO/H2O2/NO2¯ reaction systems containing increasing concentrations of KYC and measured changes in conjugated diene
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protected BAEC from cell injury from MPO released from PMA-stimulated neutrophils as well
formation.
The MPO/H2O2/NO2¯ reaction system dramatically increased LDL oxidation,
confirming reports that MPO oxidizes lipids by generating yNO2 (59, 60). Adding KYC to the MPO/H2O2/NO2¯ reaction system dose-dependently increased the lag time and decreased the overall rate of LDL oxidation. At 25 µM, KYC completely suppressed LDL oxidation (Fig. 6A). These data demonstrate that KYC is a potent inhibitor of MPO-mediated LDL peroxidation. Next, we compared the effects of KYC with those of KFC and KYS (Fig. 6B). While KYC (25 µM) completely ablated LDL oxidation, KFC increased the lag phase and delayed, but did not totally inhibit, LDL oxidation. On the basis of these findings, we reasoned that limited effects of
KYS gave a totally different inhibition profile. Instead of inhibiting LDL oxidation, KYS actually accelerated LDL oxidation (Fig. 6B). These data are consistent with the idea that the Tyr enters the active site of MPO, becomes oxidized to generate a Tyry which then accelerates LDL oxidation. Figure 6C compares the inhibitory effects of KYC with those of Tyr, Trp and GSH on LDL oxidation. In agreement with a report by others (44), Tyr (dash line) accelerated and enhanced LDL oxidation. Although findings by others (36) and our data (Fig. 1F) show that Trp effectively inhibited MPO-catalyzed HOCl production, adding Trp to MPO/H2O2/NO2¯ reaction system did not inhibit but actually enhanced LDL oxidation (Fig. 6C). Even though initial rates of LDL oxidation in MPO/H2O2/NO2¯ reaction systems were reduced by GSH (i.e., an increase in lag time), this delay was quickly lost with what appears to be an increase in thiol oxidation. Regardless, for all practical purposes GSH was ineffective for inhibiting LDL oxidation compared with KYC. Additional insight into the mechanisms by which KYC inhibited MPO-mediated LDL oxidation was gained by repeating the MPO/H2O2 oxidation studies in the absence of NO2¯. In these studies, we observed that Tyr (Fig. 6D, dash line) and Trp (Fig. 6D, dot line) increased MPO-mediated LDL oxidation, which agrees with the fact that both Tyry and Trpy, formed by
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KFC on LDL oxidation were probably the result of direct yNO2 scavenging via the thiol of KFC.
MPO oxidation, are potent oxidants that accelerate LDL oxidation. In contrast, KYC and GSH did not increase LDL oxidation. GSH is reported to be a poor substrate for MPO/H2O2 systems whose rate of reaction is on the order of 10-100 M-1s-1 (32). In addition, the glutathionyl radical that would be formed is a poor oxidant. Thus, in the presence of GSH the MPO/H2O2 system was unable to oxidize LDL. In contrast, the Tyr in KYC can be rapidly oxidized to Tyry by MPO. The fact that the MPO/H2O2 system failed to increase LDL oxidation in the presence of KYC is consistent with the fact that the Tyry was rapidly scavenged by the thiol of Cys via intramolecular electron transfer.
effects of KYC on MPO-dependent MDA formation in LDL we incubated LDL (0.5 mg/mL) in a MPO/H2O2/NO2¯ reaction system (50 nM, 50 μM, 50 μM, respectively) and measured changes in MDA formation.
Increasing concentrations of KYC dose-dependently inhibited MPO-
mediated MDA formation in LDL (Fig. 7E) just as it did conjugated diene formation (Fig. 7A). The Mechanism of KYC Inhibition. To probe the mechanism for KYC inhibiting MPO activity, first, we analyzed the changes in MPO spectra (61) after incubation with KYC and H2O2. Native MPO has absorbance peaks at 429 and 575 nm. Incubating MPO with KYC induced a red shift in the 429 nm peak and formed a new peak around 627 nm. The 575 nm peak were also slightly decreased. Including of catalase (500 U/0.5 mL) in the reaction did not eliminate the peak at 627 nm (data not shown). This result indicates that KYC directly binds to the active site of MPO and interacts with the iron-heme center (Fig. 7A, panel a). Incubation of KYC/MPO with H2O2 decreased absorbance at 429 nm and 575 nm, indicating that H2O2 activated MPO, thereby decreasing native MPO. At the same time a major increase in absorbance at 456 nm and an increase in a broad band around 627 nm were observed indicating that KYC increases the accumulation of Compound II (Fig. 7A, panel b). Taken together, these spectra clearly indicate that KYC enters the active site of MPO and binds close to the iron-heme site. Experiments were also performed in the present of methionine (1 mM). The results show no
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Malondialdehyde (MDA) is another biomarker of lipid peroxidation. To determine the
difference in the presence or absence of methionine (data not shown). In this way, KYC is able to rapidly react with compound I to form compound II. We repeated the same experiment in the presence of 150 mM NaCl (Fig. 7A, panel c and d). The results show the same trend as the reaction without NaCl although the changes were smaller, suggesting that KYC might compete against Cl− for the active site. Finally, we determined if KYC reacts with compound II directly. MPO was incubated with an excess amount of H2O2 to convert native MPO into compound II. After removing excess H2O2 with catalase, adding KYC to the reaction mixture caused a blue shift of 456 nm band and an increased absorbance at 575 nm, indicating that KYC reduced
similar to what was observed when KYC was incubated with MPO (Fig. 7A, panel a). Taken together these data suggest that KYC reacts with both compound I and II; however, the rate for KYC reacting with compound I is faster than it is for compound II. Previous studies have shown that most MPO inhibitors disrupt MPO catalytic cycles. For example, irreversible inhibitors such as azide, hydrazide, etc, are known to modify the heme to destroy the catalytic function of MPO, while indole compounds react with compound I but not compound II. All these inhibition mechanisms decrease MPO-dependent H2O2 consumption. Here we propose that KYC competes against MPO substrates by rapidly reacting with both compound I and II, which is a mechanism that is different from the mechanisms of other MPO inhibitors.
To determine if this mechanism increases H2O2 consumption as Tyr (62), we
incubated KYC with MPO and H2O2 in the presence of chloride and measured H2O2 consumption (63). Our results show that incubation of MPO with H2O2 and Cl¯ induced H2O2 consumption and that addition of KYC dose-dependently increased H2O2 consumption (Fig. 7B). As KYC inhibits HOCl formation, this increase in H2O2 consumption once again suggests that KYC directly reacts with both compound I and II. Additional evidence supporting the idea that KYC enters the active site of MPO and reacts with compound I and II comes from docking studies. We simulated the binding of KYC in
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compound II to native MPO. At the same time a new narrow band at 627 nm formed which was
the active site of MPO using a published X-ray crystallographic structure of MPO (PDB ID: 3ZS1). Figure 7C shows that KYC (red) fits in the active site without clashing with MPO. This simulation shows that KYC can be oriented in such a way that phenolic group of tyrosine is above the iron-heme of MPO (Fig. 7C, blue molecule) with the shortest distance of 2.9 Å, which is similar to that which was reported for docking indole derivatives (64). Both Cys and Tyr of KYC point toward the opening of the pocket of the active site of MPO in such a way that the amino group of the Lys side chain in KYC interacts with carboxyl group of Glu116 of MPO (Fig. 7C, green lines) and might form salt bridge (distance 3.2 Å). Thus, the Lys helps orient KYC in
KYC binds into the active site of MPO and directly interacts with iron-heme site. KYC Inhibits MPO-dependent Lipoprotein Oxidation, Nitration and Chlorination MPO activity increases Trp oxidation, Tyr nitration and chlorination in a variety of proteins (66, 67). To determine the effect of KYC on MPO-dependent Trp oxidation in LDL, we incubated LDL with MPO/H2O2/NaNO2 for 30 min at room temperature and then measured Trp oxidation in LDL via changes in Trp intrinsic fluorescence. MPO induced significant decreases (15%, p
